Information on EC 1.3.2.3 - L-galactonolactone dehydrogenase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.3.2.3
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RECOMMENDED NAME
GeneOntology No.
L-galactonolactone dehydrogenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
L-galactono-1,4-lactone + 2 ferricytochrome c = L-ascorbate + 2 ferrocytochrome c + 2 H+
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
redox reaction
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reduction
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Ascorbate and aldarate metabolism
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Biosynthesis of secondary metabolites
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L-ascorbate biosynthesis I (L-galactose pathway)
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Metabolic pathways
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NIL
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ascorbate metabolism
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SYSTEMATIC NAME
IUBMB Comments
L-galactono-1,4-lactone:ferricytochrome-c oxidoreductase
This enzyme catalyses the final step in the biosynthesis of L-ascorbic acid in higher plants and in nearly all higher animals with the exception of primates and some birds [5]. The enzyme is very specific for its substrate L-galactono-1,4-lactone as D-galactono-gamma-lactone, D-gulono-gamma-lactone, L-gulono-gamma-lactone, D-erythronic-gamma-lactone, D-xylonic-gamma-lactone, L-mannono-gamma-lactone, D-galactonate, D-glucuronate and D-gluconate are not substrates [5]. FAD, NAD+, NADP+ and O2 (cf. EC 1.3.3.12, L-galactonolactone oxidase) cannot act as electron acceptor [5].
CAS REGISTRY NUMBER
COMMENTARY hide
9029-02-1
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
variety reticulatus
Uniprot
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
cultivar West Virginia 106
Uniprot
Manually annotated by BRENDA team
tuber cultivars Agata, Altesse, Franceline, Manon and Monalisa
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
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GLDH catalyzes the last step of L-ascorbate, AsA, biosynthesis in plants, but the upstream genes in the AsA biosynthetic pathway are responsible for enhancing the AsA content in plants, regulation, overview
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
D-arabinono-1,4-lactone + ferrocytochrome c + H+
?
show the reaction diagram
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L-galactono-1,4-lactone + 1,4-benzoquinone
L-ascorbate + reduced 1,4-benzoquinone
show the reaction diagram
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-
-
-
?
L-galactono-1,4-lactone + 2 ferricytochrome c
L-ascorbate + 2 ferrocytochrome c + 2 H+
show the reaction diagram
L-galactono-1,4-lactone + cytochrome c
L-ascorbate + reduced cytochrome c
show the reaction diagram
L-galactono-1,4-lactone + ferricytochrome c
L-ascorbate + ferrocytochrome c
show the reaction diagram
L-galactono-1,4-lactone + ferricytochrome c
L-ascorbate + ferrocytochrome c + H+
show the reaction diagram
L-galactono-1,4-lactone + phenazine methosulfate
L-ascorbate + reduced phenazine methosulfate
show the reaction diagram
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-
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?
L-gulono-1,4-lactone + cytochrome c
D-ascorbate + reduced cytochrome c
show the reaction diagram
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?
L-gulono-1,4-lactone + ferricytochrome c
?
show the reaction diagram
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?
L-gulono-gamma-lactone + ferricytochrome
D-ascorbate + ferricytochrome c
show the reaction diagram
L-mannono-gamma-lactone + ferricytochrome c
D-ascorbate + ferricytochrome c
show the reaction diagram
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18% activity of that with L-galactono-gamma-lactone
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?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
L-galactono-1,4-lactone + 2 ferricytochrome c
L-ascorbate + 2 ferrocytochrome c + 2 H+
show the reaction diagram
L-galactono-1,4-lactone + cytochrome c
L-ascorbate + reduced cytochrome c
show the reaction diagram
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-
-
?
L-galactono-1,4-lactone + ferricytochrome c
L-ascorbate + ferrocytochrome c
show the reaction diagram
L-galactono-1,4-lactone + ferricytochrome c
L-ascorbate + ferrocytochrome c + H+
show the reaction diagram
additional information
?
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incubation of leaf discs in a L-galactonolactone solution leads to a rapid 2 to 3fold increase in the L-ascorbate content in both GLDH-transformed and non-transformed plants in the same manner
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cytochrome c
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ferricytochrome c
flavin
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Cu2+
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strong inhibition
Zn2+
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inhibitory
additional information
Mg2+, Ca2+ and Sr2+ have no effect on activity
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5,5'-dithiobis(2-nitrobenzoic acid)
thiol-modifying agent
Acriflavine
Atebrin
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reaction with ferricytochrome inhibited, reaction with phenazine methosulfate not
H2O2
GALDH is inactivated by hydrogen peroxide due to selective oxidation of cysteine 340, located in the cap domain
L-galactono-1,4-lactone
L-galactono-gamma-lactone
lycorine
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menthofuran
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methyl viologen
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slightly inhibits
N-ethylmaleimide
o-phenanthroline
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in absence of substrate
p-chloromercuribenzoate
p-hydroxymercuribenzoic acid
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phosphate
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reaction with ferricytochrome inhibited, reaction with phenazine methosulfate stimulated
riboflavin
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reaction with ferricytochrome inhibited, reaction with phenazine methosulfate not
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
drought
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causes a substantial increase in GalLDH protein and activity in the low ascorbic acid cultivar Cooperativa Maipún, but has no effect in higher ascorbic acid cultivar Buck Chambergo
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.28
1,4-benzoquinone
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wild type enzyme, in the presence of L-galactono-1,4-lactone, in 50 mM sodium phosphate (pH 8.8), at 25°C
0.034 - 0.18
cytochrome c
10.2 - 107
D-Arabinono-1,4-lactone
0.12 - 123
L-galactono-1,4-lactone
0.12 - 3.3
L-galactono-gamma-lactone
12.4 - 18.7
L-gulono-1,4-lactone
0.026
phenazine methosulfate
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wild type enzyme, in the presence of L-galactono-1,4-lactone, in 50 mM sodium phosphate (pH 8.8), at 25°C
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
108
1,4-benzoquinone
Arabidopsis thaliana
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in the presence of L-galactono-1,4-lactone, in 50 mM sodium phosphate (pH 8.8), at 25°C
151
cytochrome c
Arabidopsis thaliana
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in the presence of L-galactono-1,4-lactone, in 50 mM sodium phosphate (pH 8.8), at 25°C
1.2 - 51
D-Arabinono-1,4-lactone
0.7 - 240
L-galactono-1,4-lactone
0.22 - 10.4
L-gulono-1,4-lactone
64
phenazine methosulfate
Arabidopsis thaliana
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in the presence of L-galactono-1,4-lactone, in 50 mM sodium phosphate (pH 8.8), at 25°C
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
16.4
L-galactono-1,4-lactone
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at 25°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.022
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in the presence of methyl viologen
0.0245 - 0.0513
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root mitochondria of transformed transgenic tobacco lines, pH not specified in the publication, temperature not specified in the publication
0.0399
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mitochondrial inner membrane fraction
3
extract of yeast expressing the recombinant enzyme
7
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cell extract, at pH 8.8
44.5
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mitochondria
76
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after purification, at pH 8.8
37000
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purified enzyme
additional information
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Arabidopsis ppr40-1 mutant which presents a strongly reduced electron flow through complex III, shows a higher activity of GLDH
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.2
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assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8 - 9.5
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for activity with cytochrome c
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
27
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assay at
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5
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enzyme before processing, isoelectric focusing
6.8
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theoretical value for recombinant His-tagged GALDH
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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AtGLDH mRNA level exhibits diurnal change. The level is low in the morning and increases during the day. Its level at 18.00 is about 2fold that in the morning at 6.00. Then the level decreases during the night until dawn of the next day. The diurnal change is regulated by light
Manually annotated by BRENDA team
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6800
immunoblotting, the pre-mature GalLDH with a mitochondrion-targeting signal
55000
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mature enzyme, SDS-PAGE
57110
predicted from the view that the FR/YA site is the cleavage point for the mature protein
58000
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SDS-PAGE, monomeric form of GLDH
58760
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predicted molecular mass of mature His-tagged enzyme
68800
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theoretical value
420000
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three distinct GLDH containing protein complexes of 850, 470, and 420 kDa are discovered using a newly developed in gel GLDH activity assay and immunoblotting. Subunits of the novel 470 and 420 kDa complexes are identified by mass spectrometry. Like the 850-kDa complex, they also include complex I subunits
470000
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three distinct GLDH containing protein complexes of 850, 470, and 420 kDa are discovered using a newly developed in gel GLDH activity assay and immunoblotting. Subunits of the novel 470 and 420 kDa complexes are identified by mass spectrometry. Like the 850-kDa complex, they also include complex I subunits
850000
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three distinct GLDH containing protein complexes of 850, 470, and 420 kDa are discovered using a newly developed in gel GLDH activity assay and immunoblotting. The 850-kDa complex represents the known smaller version of mitochondrial complex I. GLDH is shown to be attached to the membrane arm of the 850-kDa complex I
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
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GLDH forms part of the 850-kDa complex I and part of a rotein complexes of 470 and 420 kDa
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
52
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the recombinant enzyme has a half-life of 20 min at 52°C, in the presence of excess FAD the half-life at 52°C is increased to 115 min
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
solubilized very labile, but stabilized in the presence of L-galactono-gamma-lactone under aerobic conditions
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80°C, 0.3 mg per ml protein, 0.1 M potassium phosphate, pH 7.4, 10% glycerol,2 mM glutathione, 2 month
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-80°C, 50 mM sodium phosphate and 300 mM NaCl (pH 7.4), more than 12 months, 30-50% loss of activity
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-80°C, diluted protein, 0.1 M potassium phosphate, pH 7.4, 10% glycerol,2 mM glutathione, 30% loss of activity after 1 month
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activity in fresh extract, 4°C, and frozen, -20°C, is lost after 3 days
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crude membrane extracts from Arabidopsis thaliana leaves and roots are prepared
low recovery
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mitochondria from Nicotiana sylvestris leaves are prepared
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Ni-NTA agarose column chromatography and Q-Sepharose column chromatography
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partial, wild-type and recombinant from Escherichia coli
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cDNA encoding GLDH, subcloning in Escherichia coli strain HB101, and functional expression in transgenic Nicotiana tabacum leaves and roots via Agrobacterium tumefaciens LBA4404-mediated transformation under control of the CaMV 35S promoter. Despite the elevated GLDH activity, the L-ascorbate content in the leaves does not change in all lines, but increases 6-10fold in the roots of transgenic plants, overview
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expressed in Escherichia coli BL21(DE3) cells
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expressed in tobacco plants by Agrobacterium-mediated transformation under CaMV 35S constitutive promoter
expression in Escherichia coli
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expression in Saccharomyces cerevisiae
for expression in Escherichia coli BL21DE3 cells
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GLDH sense and antisense are transformed into rice callus by Agrobacterium-mediated infection (strain EHA105)
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into a pET-His6 vector for expression in Escherichia coli BL21DE3 cells
into pMAT037 and transformed into the EHA101 strain of Agrobacterium tumefaciens
overexpressed in lettuce Agrobacterium-mediated transformation
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
relative mRNA expression levels of galactono-1,4-lactone dehydrogenase shows no difference between wild type and ppr40-1 mutant plants
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C340A
mutant, insensitive toward thiol oxidation, exhibits poor affinity for L-galactono-1,4-lactone
C340S
mutant, insensitive toward thiol oxidation, exhibits poor affinity for L-galactono-1,4-lactone
E386A
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mutant, catalytically far less efficient than wild-type GALDH, Glu386 is involved in productive substrate binding
E386D
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mutant, catalytically far less efficient than wild-type GALDH, Glu386 is involved in productive substrate binding
L56A
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less active than the wild type enzyme
L56C
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less active than the wild type enzyme
L56F
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less active than the wild type enzyme
L56H
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less active than the wild type enzyme and releases its FAD cofactor more easily than wild type GALDH
L56I
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the mutant displays a higher turnover rate with L-galactono-1,4-lactone than the wild type enzyme
R388A
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inactive mutant, Arg388 is crucial for the stabilization of the anionic form of the reduced FAD cofactor
R388K
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mutant, shows significant activity, Arg388 is crucial for the stabilization of the anionic form of the reduced FAD cofactor
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information